Nanopore-based analysis of protein characteristics

ABSTRACT

Methods for nanopore-based protein analysis are provided. The methods address the characterization of a target protein analyte, which has a dimension greater than an internal diameter of the nanopore tunnel, and which is also physically associated with a polymer. The methods further comprise applying an electrical potential to the nanopore system to cause the polymer to interact with the nanopore tunnel. The ion current through the nanopore is measured to provide a current pattern reflective of the structure of the portion of the polymer interacting with the nanopore tunnel. This is used as a metric for characterizing the associated protein that does not pass through the nanopore.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/517,996, filed Jul. 22, 2019 (now U.S. patent Ser. No. 10/948,454),which is a continuation of U.S. patent application Ser. No. 15/120,286,filed Aug. 19, 2016 (now U.S. patent Ser. No. 10/359,395), which is thenational stage of International Application No. PCT/US2014/067732, filedNov. 26, 2014, which claims the benefit of U.S. Provisional PatentApplication No. 61/941,919, filed Feb. 19, 2014, all of whichapplications are incorporated herein by reference in their entirety.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under R01HG005115awarded by the National Human Genome Research Institute (NHGRI) of theNational Institutes of Health. The Government has certain rights in theinvention.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application was provided inU.S. patent application Ser. No. 15/120,286, which was filed Aug. 19,2016, in lieu of a paper copy and is hereby incorporated by referenceinto the specification. The name of the text file containing thesequence listing is 56752_Sequence_final_2017-01-12.txt. The text fileis 1.34 KB; was created on Jan. 12, 2017, was previously submitted aspart of U.S. patent application Ser. No. 15/120,286; and is submittedherewith via EFS-Web.

BACKGROUND

The ability to provide a fine-scale characterization of proteinconformation and movement can provide a wealth of information regardingthe protein's function. Several techniques have been developed toprovide a great advancement in resolution of such functional proteinstudies. Assays that incorporate Forster Resonance Energy Transfer(FRET) provide detectable signals when moieties attached topredetermined protein domains interact within a spatial range. However,FRET signals are generated in bulk assays that aggregate signals from alarge number of individual interactions and, thus, are inherentlylimited in resolution. Other assays avoid the data scatter inherent tobulk assays by addressing the interactions of single-molecules. Forexample, commonly used tools to conduct measurements on motor enzymesinclude optical tweezers, magnetic tweezers, tethered particle assays.For example, optical tweezers employ a highly focused laser beam to hold(or repulse) an object, such as a bead. The bead can be attached to apolymer that functions as a tether. The polymer can then be manipulatedby a target enzyme that interacts (i.e., applies force) to the polymer.These manipulations are detected by measuring the displacement of thebead (or other object) from the field applied by the laser. To date,optical tweezers can achieve a precision of ˜0.3 nm spatial resolutionat ˜1 ms time scales without ensemble averaging. The limitation of thisresolution is due, in part, to the long tether of the polymer requiredto avoid damaging the target protein by the applied laser.

The ability to observe the mechanistic functioning of complexbio-molecules directly, and not just via the input and output of bulkassays, can accelerate health care and address how biological systemsreally work. However, notwithstanding the advances of single-moleculetechniques, a need remains for inexpensive and facile techniques thatcan address mechanistic movements and conformation states of proteins atimproved spatial and temporal resolutions.

SUMMARY

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This summary is not intended to identify key features ofthe claimed subject matter, nor is it intended to be used as an aid indetermining the scope of the claimed subject matter. This summary isprovided to introduce a selection of concepts in a simplified form thatare further described below in the Detailed Description. This summary isnot intended to identify key features of the claimed subject matter, noris it intended to be used as an aid in determining the scope of theclaimed subject matter.

In one aspect, this disclosure provides a method of characterizing aprotein in a nanopore system. The nanopore system comprises a nanoporedisposed in a membrane that separates a first conductive liquid mediumfrom a second conductive liquid medium, wherein the nanopore comprises atunnel that provides liquid communication between the first conductiveliquid medium and the second conductive liquid medium, and wherein theprotein is physically associated with a polymer in the first conductiveliquid medium. The method comprises:

-   -   (a) applying an electrical potential between the first        conductive liquid medium and the second conductive liquid medium        to cause the polymer to interact with the nanopore tunnel,        wherein at least one dimension of the protein exceeds a diameter        of the nanopore tunnel;    -   (b) measuring an ion current through the nanopore during the        interaction of the polymer with the nanopore tunnel to provide a        current pattern;    -   (c) determining a position and/or movement of at least one        polymer subunit in the nanopore tunnel from the current pattern;        and    -   (d) associating the position and/or movement of the at least one        polymer subunit with a characteristic of the protein.

In one embodiment, the polymer is a nucleic acid, PNA, or a combinationthereof. In one embodiment, the nucleic acid is DNA, RNA, or acombination thereof. In one embodiment, the nucleic acid comprises anabasic residue. In one embodiment, the nucleic acid is not ahomopolymer.

In one embodiment, the protein is an enzyme. In one embodiment, theenzyme is a molecular motor. In one embodiment, the molecular motor is atranslocase, a polymerase, a helicase, an exonuclease, a viral packagingmotor, or a topoisomerase. In one embodiment, the enzyme is a Brownianmotor, Brownian ratchet ribosome, myosin, or kinesin. In one embodiment,the protein is a mutant protein or fusion protein. In one embodiment,the protein comprises two or more domains capable of mutual interaction.In one embodiment, the protein is covalently coupled to the polymer.

In one embodiment, the position and/or movement of the at least onepolymer subunit can be resolved to about 35 pm. In one embodiment, theposition of the at least one polymer subunit is associated with aconformational state of the protein. In one embodiment, the movement ofthe at least one polymer subunit is associated with a length of adiscrete translocation step of the polymer within the nanopore tunnelthat is conferred by the molecular motor. In one embodiment, themovement of the at least one polymer subunit is associated with atemporal duration of a discrete translocation step of the polymer withinthe nanopore tunnel that is conferred by the molecular motor. Thetemporal duration can be resolved to about 800 ns. In one embodiment,the movement of the at least one polymer subunit is associated with anincidence rate of polymer translocation missteps committed by themolecular motor. In one embodiment, the characteristic of the enzyme isa presence or degree of modulation of enzyme activity conferred by areaction condition or putative agonist, antagonist, or co-factor.

In one embodiment, the nanopore is a solid-state nanopore, a proteinnanopore, a hybrid solid state-protein nanopore, a biologically adaptedsolid-state nanopore, or a DNA origami nanopore. In one embodiment, theprotein nanopore is alpha-hemolysin, leukocidin, Mycobacterium smegmatisporin A (MspA), outer membrane porin F (OmpF), outer membrane porin G(OmpG), outer membrane phospholipase A, Neisseria autotransporterlipoprotein (NalP), WZA, Nocardia farcinica NfpA/NfpB cationic selectivechannel, lysenin or a homolog or variant thereof. In one embodiment, theprotein nanopore sequence is modified to contain at least one amino acidsubstitution, deletion, or addition. In one embodiment, the at least oneamino acid substitution, deletion, or addition results in a net chargechange in the nanopore. In one embodiment, the protein nanopore has aconstriction zone with a non-negative charge.

In one embodiment, the electrical potential applied is between 10 mV and1 V or between −10 mV and −1 V.

In another aspect, the disclosure provides a method of characterizing aprotein in a nanopore system. The comprises a nanopore disposed in amembrane that separates a first conductive liquid medium from a secondconductive liquid medium, wherein the nanopore comprises a tunnel thatprovides liquid communication between the first conductive liquid mediumand the second conductive liquid medium, and wherein the protein isphysically associated with a polymer in the first conductive liquidmedium. The method comprises:

-   -   (a) applying an electrical potential between the first        conductive liquid medium and the second conductive liquid medium        to cause the polymer to interact with the nanopore tunnel,        wherein at least one dimension of the protein exceeds a diameter        of the nanopore tunnel;    -   (b) measuring an ion current through the nanopore during the        interaction of the polymer with the nanopore tunnel to provide a        first current pattern;    -   (c) comparing the first current pattern to a reference current        pattern;    -   (d) determining a change in position and/or movement of at least        one polymer subunit in the nanopore tunnel from the position        and/or movement of at least one polymer subunit in the nanopore        tunnel determined from the reference current pattern; and    -   (e) associating the change in position and/or movement of the at        least one polymer subunit in the nanopore tunnel with a        characteristic of the enzyme.

In one embodiment, the nanopore system comprises a difference from thenanopore system used to generate the reference current pattern. In oneembodiment, the difference is the presence or absence of a putativeprotein agonist, antagonist, or co-factor in the first conductivemedium. In one embodiment, the difference is a difference concentrationof a putative protein agonist, antagonist, or co-factor in the firstconductive medium. In one embodiment, the characteristic is a presenceor degree of modulation of protein activity or conformation conferred bythe putative agonist, antagonist, or co-factor. In one embodiment, thedifference is at least one amino acid difference in the amino acidsequence of the protein compared to the amino acid protein sequence inthe nanopore system used to generate the reference current pattern. Inone embodiment, the characteristic is a presence or degree of modulationof protein activity or conformation conferred by the amino aciddifference in the amino acid sequence. In one embodiment, the methodfurther comprises generating a reference current pattern.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1A is a cartoon illustration of an exemplary nanopore system usefulin the practice of the present disclosure. Schematically, a singlenanopore (e.g., MspA) is embedded in a phospholipid bilayer thatseparates two volumes of conductive liquid media, such as an electrolytemixture. A voltage across the bilayer causes an ion current to flowthrough the interior of the nanopore. A protein, such as a molecularmotor enzyme, is physically associated with a polymer (e.g., DNA) thatis drawn to the interior of the nanopore. The single stranded end passesinto and through the nanopore until the protein, which exceeds thelargest diameter of the interior tunnel of the nanopore, comes to reston the pore. The ion current in the nanopore tunnel is influenced by thenucleotide structures (thus identity) within the narrowest portion ofthe nanopore tunnel (“constriction”).

FIG. 1B is a graphical illustration of a representative current patternproduced by a nanopore system using an MspA nanopore and DNA associatedwith a phi29 DNA polymerase enzyme. The current pattern indicates thatthe DNA is moved through the nanopore by the phi29 DNA polymerase (DNAP)enzyme in discrete translocation steps. The observed current levels canbe associated with DNA sequence. Occasional back-stepping activity ofthe phi29 DNAP causes repetitions of levels indicated by *.

FIG. 1C is a graphical representation of the time-ordered mean ioncurrent values derived from the original, stochastic level durations.

FIGS. 2A-2F illustrate the process and sensitivity of the PINT system bycharacterizing at a small scale the DNA movement in the nanopore forprotein analysis.

FIG. 2A graphically illustrates the current levels (solid black lines)corresponding to the shown DNA sequence sequence (set forth as SEQ IDNO:1), which provide a distance measure (in nt). A spline profile(curved line) is used to demonstrate distances in between levels. Thestandard deviation of the current levels yield the precision to whichdistances can be measured. X in the indicated sequence represents anabasic residue. FIG. 2B is a cartoon illustration of DNA moving withinthe constriction of MspA by a distance δ. FIG. 2C graphicallyillustrates current levels corresponding to the shown DNA sequence (setforth as SEQ ID NO:2) observed from the nanopore system at 180 mV(circles), and at 140 mV (triangles). FIG. 2D graphically illustratescurrent values for 180 mV (circles) and a spline fit to those levels(dotted curve). Triangles present the levels taken at 140 mV, asillustrated in FIG. 2C, after a multiplicative scale and additiveoffset. For the scaled 140 mV levels, the horizontal position isdisplaced by 0.3 nt to put the levels in-line with the spline profilefor the current levels observed at 180 mV. This indicates the appliedvoltage shifts the DNA within MspA by 0.3 nt. FIG. 2E illustrates thecorresponding observed time-ordered mean ion current levels derived fromthe original current pattern where the DNA was moved by phi29 DNAP. Thelevels correspond to the DNA sequence (set forth as SEQ ID NO:3) andhence, the physical displacement of the DNA sequence relative to MspA. Adashed line overlays current levels to indicate the current profilecorresponding to the specific DNA sequence when moving continuouslythrough the nanopore. FIG. 2F graphically illustrates the observedtime-ordered mean ion current levels derived from the original currentpattern where the same DNA (set forth as SEQ ID NO:3) was moved byhel308 TGA. When DNA motion is controlled by the translocase activity ofhel308 TGA, a level profile directly comparable to that generated by DNAtranslocation controlled by phi29 DNAP is observed. However, the hel308TGA current pattern shows twice as many levels for the same DNAsequence. This suggests that hel308 TGA moves DNA twice per nucleotide,relative to MspA.

FIGS. 3A-3G illustrate the nanopore-based analysis of DNA translocationsteps controlled by hel308. The DNA sequence in FIG. 3A is set forth asSEQ ID NO:4.

FIG. 3A graphically illustrates a consensus current level patterngenerated in a nanopore system for a DNA polymer associated with phi29DNA polymerase.

FIG. 3B graphically illustrates a consensus current level patterngenerated in the same nanopore system for the same DNA polymer as inFIG. 3A, but where the DNA is associated with the helicase hel308 TGA.Each one-nucleotide translation along the DNA is divided into twodistinct steps, compared to FIG. 3A. All helicase data are taken at theexperimental conditions of 22° C., 300 mM KCl, 5 mM MgCl₂ and 180 mV.

FIG. 3C graphically illustrates the half-life of current levelsindicated in FIG. 3B. The level duration alternates between long andshort durations. The duration of every other level is dependent on ATPconcentration (“[ATP]”), as determined by using different concentrationsof ATP: 10 μM ATP (dashed lines) and 1000 μM ATP (solid lines).

FIG. 3D graphically illustrates that the difference of the durationswith high and with low [ATP] removes sequence dependence that alsoinfluences the step durations.

FIG. 3E graphically illustrates the average durations of levels versus[ATP]⁻¹ for ATP-independent steps (long dashed lines) and theATP-dependent steps (short dashed lines). For the ATP-independent stepswe measured an average rate of 4.5+/0.4 s⁻¹. For the ATP-dependentsteps, we observed Michaelis Menton kinetics with a maximum velocity of15.2+/−1.3/s and the Michaelis constant of 92.5+/−9.9 μmol.

FIG. 3F graphically illustrates that half-life of levels depend on theidentity of the nucleotide that had passed through the constriction:A=alternating long-short dash, C=solid, G=light, short dash, T=heavy,long dash; the lower set of lines represent the ATP-dependent steps andupper set represent the ATP-independent steps with [ATP] at 500 μM. Thepeaks at 14, 17, and 18 indicate positions located within the enzymewhere G, T, or C's, respectively, cause longer level durations.

FIG. 3G graphically illustrates the phase of steps relative to the phi29DNAP. The ATP-independent steps are represented as solid bars and theATP-dependent steps are represented in open bars. The average hel308step length between ATP-independent and ATP-dependent steps is 0.53±0.04nt. Average uncertainties are standard deviations of the mean. The ioncurrent uncertainties for the levels means illustrated in FIG. 3A are,on average smaller, than the line width, <0.1 pA.

DETAILED DESCRIPTION

The present disclosure relates to the inventors' advancements to theanalysis and characterization of target proteins using nanopore basedsystems.

Nanopore systems have been previously employed to characterize a varietyof analytes, such as small molecules and polymers. These methodsgenerally involve passing the target analyte through a nanoscopicopening while monitoring a detectable signal, such as an electricalsignal. The signal is influenced by the physical properties of thetarget analyte as it passes through the nanopore and, thus, can beassociated with a structural feature of the analyte, such as itsidentity. When addressing polymeric analytes, for example,single-stranded DNA (“ssDNA”), the discrete detectable signals can beinfluenced by the structure of each consecutive polymer subunit when thepolymer passes linearly through the nanopore, thus providing informationregarding the sequence of the polymer.

The present inventors have co-opted the above nanopore system approachto investigate larger analytes that are unable to pass through thenanopore opening, instead of small analytes that can enter the interiorspace of the nanopore. As described in more detail below, the inventorsdiscovered that important features of larger protein analytes can becharacterized using nanopore systems, notwithstanding the fact that theydo not pass through the nanopore. Briefly stated, in this novel approacha polymer is associated with the target protein. As the polymerinteracts with the interior of the nanopore tunnel, the associatedprotein is pulled toward the opening rim of the nanopore but cannot passthrough due to its size. Using this information, the polymer can now beused as a measurement tool to ascertain a distance between the nanoporeconstriction zone and the target protein to a resolution as small as30-40 picometers. Furthermore, the polymer-protein association need notbe static, but can be dynamic. In this case, the polymer's movementsthrough the nanopore can be monitored in real time with a resolutionshorter than a millisecond, such as to about 700 microseconds or 800microseconds. Accordingly, a wide variety of protein characteristics canbe investigated at spatial and temporal resolutions heretofore unseen inexisting technologies, such as with molecular tweezers and FRETanalysis. As will be discussed, the nanopore system can be configured toaddress a wide variety of protein characteristics, such as the nature offolding and conformational changes, the structural and conformationaleffects of mutations in protein sequence, and the nature of molecularmotor-polymer interactions. Moreover, these experimental configurationscan be applied to broader investigations of potential drug panels andtheir effects on the activity of enzymes, such as molecular motors, andthe like. These and other advantages and applications will become moreapparent in view of the below description.

In one aspect, the present disclosure provides a method ofcharacterizing a protein in a nanopore system. In this method, theprotein is physically associated with a polymer. The method comprisesthe steps of: (a) applying an electrical potential between the firstconductive liquid medium and the second conductive liquid medium of thenanopore system to cause the polymer to interact with the nanoporetunnel; (b) measuring an ion current through the nanopore during theinteraction of the polymer with the nanopore tunnel to provide a currentpattern; (c) determining a position and/or movement of at least onepolymer subunit in the nanopore tunnel from the current pattern; and (d)associating the position and/or movement of the at least one polymersubunit with a characteristic of the protein.

Various aspects of nanopore systems encompassed by the presentdisclosure are described in more detail below. Generally described, thenanopore system comprises a nanopore disposed in a membrane thatseparates a first conductive liquid medium from a second conductiveliquid medium. The nanopore generally forms an interior tunnel thatprovides liquid communication between the first conductive liquid mediumand the second conductive liquid medium. In the present aspect, theprotein is disposed in the first conductive liquid medium and isphysically associated with the polymer.

As used herein, the term “physically associated” can refer to a covalentbond to provide a permanent or static association between the proteinand the polymer. Alternatively, the term can refer to a non-covalentbond or association between the protein and the polymer. Thisencompasses embodiments where the protein can have a dynamic physicalassociation with the polymer, such as in the case of many molecularmotor enzymes that can contact and apply force to polymer molecules(e.g., nucleic acids) and may move along the length of the polymer in adynamic movement.

In this method, at least one dimension of the protein exceeds a diameterof the nanopore tunnel. Accordingly, any movement of the associatedpolymer into the interior space of the nanopore does not result in thepassage of the protein itself through the nanopore. Instead, the proteinis merely pulled into contact with the outer rim entrance of thenanopore and comes to rest at the outer rim of the nanopore with nofurther progression towards the opposite side of the membrane. Thus, theprotein provides an anchor, whether dynamic or substantially static, tothe polymer, that provides resistance to further movement of the polymerinto (and possibly through) the nanopore. Thus, by virtue of theprotein's position of the protein at the outer rim entrance of thenanopore, the protein's association with the polymer results in acontrolled rate of polymer movement (or a substantial prevention offurther movement) into or through the nanopore.

The protein is the target analyte for the present disclosure. It will beappreciated that the present disclosure can be widely applied to anytarget protein of interest for a wide variety of assays. Thus, thepresent disclosure is not limited to a particular target protein-type.The two limitations are that the protein must have at least onedimension that exceeds an internal diameter of the nanopore to preventpassage of the protein through the nanopore (described above) and thatthe protein must be capable or amenable to a physical association to thepolymer. It will be appreciated that the protein can be any naturallyoccurring protein, any modified (e.g., engineered) protein, includingmutated or fusion proteins. Several categories of potential proteinswill be described, although it is noted that these descriptions aremerely for illustration purposes and are not intended to be limiting.

In some embodiments, the protein is an enzyme. Broadly defined, andenzyme is a polypeptide macromolecule that, when properly folded into atertiary structure, can perform work such as catalyze a reaction.

In some embodiments, the enzyme is a molecular motor. A “molecularmotor” is broadly defined as a protein, such as an enzyme, thatinteracts with a particular polymer, such as a nucleic acid. In someembodiments, the interaction involves some force applied to the polymer.In a natural situation, the force might result in the attachment of themolecular motor to the polymer, movement of the molecular motor alongthe polymer, or a change in conformation or shape of the polymer. Theforce can result in the manipulation of the polymer, such as causing themovement of the polymer in the nanopore system. The molecular motor canbe active, i.e., using energy such as ATP to move or interact with thepolymer. Such molecular motors can encompass moieties that can move thepolymer against the force direction applied by the voltage cross thenanopore. Alternatively, the molecular motor can be passive, i.e., notusing energy to move or interact with the polymer. The presentdisclosure is useful to characterize the nature of the associationbetween the molecular motor and the particular polymer. For example,many molecular motors move along a nucleic acid strand in discrete andrepetitive steps. Such molecular motors, when immobilized against theouter rim entrance of the nanopore, facilitate movement of the nucleicacid in discrete steps through the nanopore in a stepwise fashion wherethe nucleic acid progresses in discrete movements of a relativelyconsistent length, akin to a ratchet or queuing motion. Some molecularmotors, such as phi29 DNA polymerase (DNAP), move the nucleic acidpolymers in single measurable nucleotides steps through the nanopore.However, it will be appreciated that other molecular motors are usefulfor moving the nucleic acid polymers in steps that are less than asingle nucleotide length. Yet other molecular motors are useful formoving the nucleic acid polymers in steps that are more than a singlenucleotide in length.

The present method can be used to measure characteristics such as thedistance of each movement at a sub-Angstrom resolution by monitoring theresultant movement of the polymer through the nanopore. The method canalso be used to characterize the energy requirements of the molecularmotor action, by adjusting the availability of chemical energy (such asthe concentration of ATP). As another example, the putative co-factors,agonist, antagonist, or any other potential reaction condition can betested to ascertain the changes conferred on the monitored movement ofthe polymer through the nanopore. As yet another example, the method canbe applied to characterize the rate of the polymer movement through thepore facilitated by the protein in any particular reaction condition orenvironment. Moreover, molecular motors often commit mistakes whereinthe molecular motor skips a step or backs up and repeats a movementstep. Such skips or toggles can be detected in the current patterns. SeePCT/US2014/059360, incorporated herein by reference in its entirety.

Illustrative, nonlimiting examples of such molecular motors are providedbelow.

The molecular motor can be a naturally occurring enzyme, an engineeredor mutated enzyme, or otherwise derived from an enzyme. In someembodiments, the molecular motor is modified to remove a particularfunction from the enzyme, but preserves the ability of the molecularmotor to associate with the polymer analyte (e.g., nucleic acid) andfacilitate its movement within the nanopore. In some embodiments, theenzyme is or is derived from a member of any of the EnzymeClassification (EC) groups 3.1.11, 3.1.13, 3.1.14, 3.1.15, 3.1.16,3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30 and 3.1.31. The enzymemaybe any of those disclosed in International Publication No. WO2010/1086603, incorporated herein by reference in its entirety.

In some embodiments, the enzyme is a translocase, a polymerase, ahelicase, an exonuclease, or topoisomerase, and the like.

Many exemplary exonucleases are generally described in WO 2010/1086603,incorporated herein by reference in its entirety. Other examples areexonucleases, which can include exonuclease I, exonuclease III, lambdaexonuclease, or a variant or homolog thereof. For any aspect herein,homologs, derivatives, and other variant proteins, as described herein,can preferably be at least 50% homologous to the reference protein basedon amino acid sequence identity. More preferably, the variantpolypeptide may be at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90% and morepreferably at least 95%, 97%, or 99% homologous based on amino acididentity to the reference protein, or any range derivable therein.Homology can be determined by any method accepted in the art. Thus,homologs or variants can possess sequence and structural modifications.The present disclosure can be useful to determine or otherwisecharacterize the functional similarities and/or differences that resultfrom the indicated differences. While exonucleases often containenzymatic functions for excising portions of the nucleic acids, suchenzymes can be modified to ablate such nuclease function whilepreserving the ability to bind and move the nucleic acid polymer.

Exemplary helicases that can be target proteins are generally describedin WO 2014/013260 and WO 2013/057495, each reference incorporated hereinby reference in its entirety, and can include a hel308 helicase, a RecDhelicase, a Tral helicase, a Tral subgroup helicase, an XPD helicases,or a variant or homolog thereof.

Exemplary polymerases that can be target proteins include DNApolymerases such as phi29 DNA polymerase (sometimes referred to as phi29DNAP), Klenow fragment, or a variant or homolog thereof.

Exemplary topoisomerases can include a gyrase, or a variant or homologthereof.

Other target proteins include viral packaging motors, or any other viralor pathogen enzyme that facilitates invasion, replication, or otherpathogenic function by the pathogen.

Yet other exemplary target proteins include Brownian motors, Brownianratchet ribosome, myosin, kinesin, and the like, as are known in theart.

As indicated above, the present method can also be applied tocharacterize conformational states of proteins. In this regard, thewildtype target protein need not have any affinity for associating withthe polymer. Instead the polymer can be covalently coupled to theprotein according to any standard and commonly recognized technique inthe art. In this context, the conformational state can be characterizedby the position of a particular polymer subunit within the nanopore.This is indicative of the distance between the protein and theparticular polymer subunit, or indeed the constriction zone of thenanopore. Any change in this conformational state can result in minutechanges in this distance, which are detectable in this system. Thus,multiple proteins can be compared (using the same polymer-type attachedin the same manner, i.e., to the same amino acid residue of theprotein). This permits mutational studies to characterize theconformational changes that result from the introduction of one or moremutations into a protein sequence. Additionally, a protein may be anatural or fusion protein that comprises two or more domains thatmutually interact, thus causing a conformational change. The variousparameters of this interaction can be inferred by measuring the movementof the polymer in the nanopore, such as the frequency, duration, andquality (inferred by distance of polymer movement).

In the present method, the application of an electrical potential acrossthe membrane (i.e., between the first conductive liquid medium and thesecond conductive liquid medium) causes the polymer to interact with thenanopore tunnel. Typically, the polymer analyte (e.g., nucleic acid)interacts with the nanopore tunnel in a linear fashion where the polymeris extended linearly along the axis of the nanopore tunnel. In someembodiments, this axis is transverse to the membrane. The term“interact,” when used with respect to the nanopore tunnel, indicatesthat the polymer moves into at least an interior portion of the nanoporeto an extent that the presence of the polymer influences the measurableion current that runs through the nanopore tunnel. As described in moredetail below, many nanopores have a “constriction” or “constrictionzone,” which is an area of the internal tunnel that has the smallestdiameter and, thus, where the current is most likely to bedifferentially affected by the presence of varying polymer structures.

The polymers encompassed by this disclosure can be any polymer capableof 1) an association with the target protein, and 2) an interaction withthe interior tunnel of the nanopore such that an ionic current throughthe nanopore can be measurably affected by the structure of the polymer.In practice, the polymer serves as a yardstick to characterize distancebetween the protein, to which the polymer is attached and is situated atthe outer rim opening of the nanopore, and the region within the tunnelwhere the presence of the polymer can affect the measurable currentwithin the pore (often referred to as the “constriction zone”).Measurement of this distance is possible because the position of polymersubunits can be monitored within the nanopore due to the variations inthe current pattern observed during the assay. The determination of theposition within a nanopore of a polymer nucleotide subunit in a nucleicacid polymer is described in more detail in PCT/US2014/059360,incorporated herein by reference in its entirety.

As used herein, a “polymer” refers to any macromolecule that comprisestwo or more linear units (also known as a “mers” or “subunits”), whereeach subunit may be the same or different. Non-limiting examples ofpolymers encompassed by the present disclosure include nucleic acids,peptides, and proteins, as well as a variety of hydrocarbon polymers(e.g., polyethylene, polystyrene) and functionalized hydrocarbonpolymers, wherein the backbone of the polymer comprises a carbon chain(e.g., polyvinyl chloride, polymethacrylates). The term “polymer” canalso include copolymers, block copolymers, and branched polymers such asstar polymers and dendrimers.

In any embodiment, there is no requirement that the polymer sequence beknown a priori, or even be decipherable from the current patternsproduced in the nanopore system. Instead, among the polymer subunits ameasureable change in the ion current can be produced and the positionof the structural variation in the polymer be ascertainable relative tothe nanopore tunnel and/or the position of the protein at the outerentrance rim. Accordingly, in some embodiments the polymer (e.g.,nucleic acid) is not a homopolymer.

The term “nucleic acid” refers to any polymer molecule that comprisesmultiple nucleotide subunits (i.e., a polynucleotide). Nucleic acidsencompassed by the present disclosure can include deoxyribonucleotidepolymer (DNA), ribonucleotide polymer (RNA), cDNA or a synthetic nucleicacid known in the art, such as peptide nucleic acid (PNA), glycerolnucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid(LNA) or other synthetic polymers with nucleotide side chains, or anycombination thereof. The nucleic acids can be in either single- ordouble-stranded form, or comprise both single and double strandedportions. Typically cDNA, RNA, GNA, TNA, or LNA are single stranded. DNAcan be either double stranded (dsDNA) or single stranded (ssDNA).

Nucleotide subunits of the nucleic acid polymers can be naturallyoccurring or artificial or modified. A nucleotide typically contains anucleobase, a sugar, and at least one phosphate group. The nucleobase istypically heterocyclic. Suitable nucleobases include purines andpyrimidines and more specifically adenine (A), guanine (G), thymine (T)(or typically in RNA, uracil (U) instead of thymine (T)), and cytosine(C). The sugar is typically a pentose sugar. Suitable sugars include,but are not limited to, ribose and deoxyribose. The nucleotide istypically a ribonucleotide or deoxyribonucleotide. The nucleotidetypically contains a monophosphate, diphosphate, or triphosphate. Theseare generally referred to herein as nucleotides or nucleotide residuesto indicate the subunit. Without specific identification, the generalterms nucleotides, nucleotide residues, and the like, are not intendedto imply any specific structure or identity. The nucleotides can also besynthetic or modified. For example, the nucleotide can be labeled ormodified to act as a marker with a distinct signal. Furthermore, beforethe application of the electric potential, modifications can be appliedto the nucleic acid that selectively affects the structure of a limitednucleotide-type to enhance the differentiation of the resulting signalfor the targeted residue (subunit). For example, see InternationalApplication No. PCT/US2014/53754, incorporated herein by reference inits entirety. One particular advantageous strategy for the practice ofthe present disclosure is to incorporate a nucleic acid residue with amissing base structure, for example, an abasic unit or spacer in thepolynucleotide. This is particularly advantageous because abasicresidues have been observed to result in a marked current spike (i.e.,sharp increase in current) when positioned within the constriction zone.Accordingly, the specific position of the abasic residue (or residues)can be readily monitored with little risk of signal confusion. Thisprovides a useful signal for monitoring the position and movement of theabasic residue through the nanopore, as permitted or influenced by theassociated protein.

The present disclosure also encompasses the use of polypeptides as thepolymer. A “polypeptide” is a macromolecule of multiple amino acidslinked by peptide (amide) bonds. As used herein, an “amino acid” refersto any of the naturally occurring amino acids found in proteins,D-stereoisomers of the naturally occurring amino acids (e.g.,D-threonine), unnatural amino acids, and chemically modified aminoacids. Each of these types of amino acids is not mutually exclusive.α-Amino acids comprise a carbon atom to which is bonded an amino group,a carboxyl group, a hydrogen atom, and a distinctive group referred toas a “side chain.” The side chains of naturally occurring amino acidsare well known in the art and include, for example, hydrogen (e.g., asin glycine), alkyl (e.g., as in alanine, valine, leucine, isoleucine,proline), substituted alkyl (e.g., as in threonine, serine, methionine,cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginine,and lysine), arylalkyl (e.g., as in phenylalanine and tryptophan),substituted arylalkyl (e.g., as in tyrosine), and heteroarylalkyl (e.g.,as in histidine).

The following abbreviations are used for the 20 naturally occurringcanonical amino acids: alanine (Ala; A), asparagine (Asn; N), asparticacid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu;E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine(Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M),phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine(Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

Unnatural amino acids (that is, those that are not naturally found inproteins) are also known in the art, as set forth in, for example, Mol.Cell. Biol., 9: 2574 (1989); J. Amer. Chem. Soc., 112:4011-4030 (1990);J. Amer. Chem. Soc., 56:1280-1283 (1991); J. Amer. Chem. Soc.,113:9276-9286 (1991); and all references cited therein. β- and γ-aminoacids are known in the art and are also contemplated herein as unnaturalamino acids.

As used herein, a “chemically modified amino acid” refers to an aminoacid whose side chain has been chemically modified. For example, a sidechain may be modified to comprise a signaling moiety, such as afluorophore or a radiolabel. A side chain may be modified to comprise anew functional group, such as a thiol, carboxylic acid, or amino group.Post-translationally modified amino acids are also included in thedefinition of chemically modified amino acids.

As described above, the current patterns produced in the describedsystems that contain a target protein associated with a polymer can beused to ascertain a characteristic of the protein. This is enabled bythe discovery that such nanopore systems can permit a highly resolvedinference of the position of a single polymer subunit within thenanopore, and changes in that position over minute ranges of time. Thisanalysis is based on a preliminary force spectroscopy investigation onsingle-stranded DNA (ssDNA) within a nanopore. The inventors previouslyfound that an anchored DNA analyte stretches within the constrictionzone of MspA with increasing force, as applied with an increasedelectric potential in the nanopore system. By varying electric potentialin the nanopore system and simultaneously monitoring the resultingcurrent, the stretching of the DNA within the nanopore was characterizedat angstrom-level precision. Using a freely jointed chain model toassess the stretching, the relative positions of the nucleotides werecharacterized during the stretch events and ascertaining the relativecontribution of Brownian motion to the sensitivity of the nanoporesystem to multiple nucleotides was established.

Due to the insight from the spring modeling analysis, the positions ofthe nucleotides can be calculated at any point during the DNAinteraction with the nanopore tunnel. Thus the current pattern isamenable to analysis that identifies any current pattern ascorresponding to a segment of the nucleic acid residing in theconstriction zone of the nanopore associated with the application of anelectrical potential. Thus, in some embodiments, the conversion of thecurrent-potential curve into a current-nucleic acid distance curve isaccomplished by application of a spring-based model. In someembodiments, the model is a model of spring with a linear restoringforce. In some embodiments, the model is a non-linear restoring force asin a freely jointed chain (FJC) model or modified freely jointed chain(FJC) model, as described in more detail below. Other appropriate modelscan be applied according to the skill in the art. See PCT/US2014/059360,incorporated herein by reference in its entirety.

In another aspect, the present disclosure provides a method ofcharacterizing a protein in a nanopore system. As above, the protein inthis aspect is physically associated with a polymer. The method of thisaspect specifically comprises: (a) applying an electrical potentialbetween the first conductive liquid medium and the second conductiveliquid medium to cause the polymer to interact with the nanopore tunnel;(b) measuring an ion current through the nanopore during the interactionof the polymer with the nanopore tunnel to provide a first currentpattern; (c) comparing the first current pattern to a reference currentpattern; (d) determining a change in position and/or movement of atleast one polymer subunit in the nanopore tunnel from the positionand/or movement of at least one polymer subunit in the nanopore tunneldetermined from the reference current pattern; and (e) associating thechange in position and/or movement of the at least one polymer subunitin the nanopore tunnel with a characteristic of the enzyme.

Various aspects of nanopore systems encompassed by the presentdisclosure are described in more detail below. Generally described, thenanopore system comprises a nanopore disposed in a membrane thatseparates a first conductive liquid medium from a second conductiveliquid medium, wherein the nanopore comprises a tunnel that providesliquid communication between the first conductive liquid medium and thesecond conductive liquid medium, and wherein the protein is physicallyassociated with a polymer in the first conductive liquid medium.

As described above, the at least one dimension of the protein exceeds adiameter of the nanopore tunnel.

The present parameters and features of this method are as described incontext of the method above. In this aspect, the method involvedcomparing the first current pattern to a reference current pattern.Thus, this method is applicable to an experimental setup to ascertainthe effect of one or more changes in conditions of a reaction. Theeffect is ideally attributable to a characteristic or effect on thetarget protein. Thus, when a difference is detected between the polymerpositions, as reflected in the first current pattern and a referencecurrent pattern, the difference can be attributed to a change in theassay conditions that produced each respective current pattern. Thus,the conditions of the assay in the recited nanopore system comprises aperturbation, or difference, compared to the conditions used to generatethe reference current pattern.

In some embodiments, the difference can be the addition or removal of aputative protein agonist, antagonist, or co-factor. In such embodiments,the method can be employed to test one or more of a panel of potentialfactors suspected of influencing a protein. For example, factorssuspected of potentially specifically inhibiting a viral helicase can betested and the ability of the helicase to move along DNA characterizedby measuring the rate of movement of the DNA polymer in the nanopore.

In other embodiments, mutations in the protein that are suspected ofaltering the interaction with nucleic acid polymers can be tested bycharacterizing the speed, frequency, or character of nucleic acidmovements.

In other embodiments, the difference can be a difference in reactionconditions, such as a difference in the presence of a co-factor, or analteration in the co-factor. In other embodiments, the difference can bea change in the concentration (either higher or lower) of componentslike ATP, and the like.

The first and reference current patterns can be generated in the same ordifferent nanopore system setup with the same or different protein andassociated polymer. In some embodiments, the system, protein, andpolymer are substantial duplicates but for the particular introducedperturbation. In some embodiments, the method comprises generating thereference current pattern. In some embodiments, the reference currentpattern is generated before or after the first current pattern isgenerated, wherein the patterns are each generated before or after theintroduction of the perturbation, respectively. In some embodiments, theperturbation is introduced into the system and the effect on theconformation of the protein is ascertained by ascertaining the changesin polymer (or polymer subunit) position or movement within thenanopore.

Various aspects of the nanopore systems as employed in the presentdisclosure are described below.

Nanopore-based analysis methods have previously been investigated forthe characterization of analytes that are passed through the nanopore.The systems permit the passing of a polymeric molecule, for example,single-stranded DNA (“ssDNA”), through a nanoscopic opening whileproviding a signal, such as an electrical signal, that is influenced bythe physical properties of the polymer subunits that reside in the closephysical space of the nanopore tunnel at any given time. The nanoporeoptimally has a size or three-dimensional configuration that allows thepolymer to pass only in a sequential, single file order. Undertheoretically optimal conditions, the polymer molecule passes throughthe nanopore at a rate such that the passage of each discrete monomericsubunit of the polymer can be correlated with the monitored signal.Differences in the chemical and physical properties of each monomericsubunit that makes up the polymer, for example, the nucleotides thatcompose an ssDNA, result in characteristic electrical signals that canidentify each monomeric subunit as it passes through the nanopore.Nanopores, such as solid state nanopores and protein nanopores heldwithin lipid bilayer membranes, have been heretofore used for analysisof DNA, RNA, and polypeptides and, thus, provide an advantageousplatform for a robust analysis of polymer position and movement as areflection on an associated protein.

A “nanopore” specifically refers to a pore typically having a size ofthe order of nanometres that allows the passage of analyte polymers(such as nucleic acids) therethrough. Typically, nanopores encompassedby the present disclosure have an opening with a diameter at its mostnarrow point of about 0.3 nm to about 2 nm. Nanopores useful in thepresent disclosure include any pore capable of permitting the lineartranslocation of the analyte polymer from one side to the other at avelocity amenable to monitoring techniques, such as techniques to detectcurrent fluctuations.

Nanopores can be biological nanopores (e.g., proteinaceious nanopores),solid state nanopores, hybrid solid state protein nanopores, abiologically adapted solid state nanopore, a DNA origami nanopore, andthe like.

In some embodiments, the nanopore comprises a protein, such asalpha-hemolysin, anthrax toxin and leukocidins, and outer membraneproteins/porins of bacteria such as Mycobacterium smegmatis porins(Msp), including MspA, outer membrane porins such as OmpF, OmpG, OmpATb,and the like, outer membrane phospholipase A and Neisseriaautotransporter lipoprotein (NaIP), and lysenin, as described in U.S.Publication No. US2012/0055792, International PCT Publication Nos.WO2011/106459, WO2011/106456, WO2013/153359, and Manrao et al., “ReadingDNA at single-nucleotide resolution with a mutant MspA nanopore andphi29 DNA polymerase,” Nat. Biotechnol. 30:349-353 (2012), each of whichis incorporated herein by reference in its entirety. Nanopores can alsoinclude alpha-helix bundle pores that comprise a barrel or channel thatis formed from a-helices. Suitable a-helix bundle pores include, but arenot limited to, inner membrane proteins and an outer membrane proteins,such as WZA and ClyA toxin. In one embodiment, the protein nanopore is aheteroligomeric cationic selective channel from Nocardia faricinicaformed by NfpA and NfpB subunits. The nanopore can also be a homolog orderivative of any nanopore illustrated above. A “homolog,” as usedherein, is a gene or protein from another species that has a similarstructure and evolutionary origin. By way of an example, homologs ofwild-type MspA, such as MppA, PorM1, PorM2, and Mmcs4296, can serve asthe nanopore in the present invention. Protein nanopores have theadvantage that, as biomolecules, they self-assemble and are essentiallyidentical to one another. In addition, it is possible to geneticallyengineer protein nanopores, thus creating a “derivative” of a nanopore,such as those illustrated above, that possesses various attributes. Suchderivatives can result from substituting amino acid residues for aminoacids with different charges, from the creation of a fusion protein(e.g., an enzyme+alpha-hemolysin). Thus, the protein nanopores can bewild-type or can be modified to contain at least one amino acidsubstitution, deletion, or addition. In some embodiments, the at leastone amino acid substitution, deletion, or addition results in adifferent net charge of the nanopore. In some embodiments, thedifference in net charge increases the difference of net charge ascompared to the first charged moiety of the polymer analyte. Forexample, if the first charged moiety has a net negative charge, the atleast one amino acid substitution, deletion, or addition results in ananopore that is less negatively charged. In some cases, the resultingnet charge is negative (but less so), is neutral (where it waspreviously negative), is positive (where it was previously negative orneutral), or is more positive (where it was previously positive but lessso). In some embodiments, the alteration of charges in the nanoporeentrance rim or within the interior of the tunnel and/or constrictionfacilitate the entrance and interaction of the polymer with the nanoporetunnel.

In some embodiments, the nanopores can include or comprise DNA-basedstructures, such as generated by DNA origami techniques. Fordescriptions of DNA origami-based nanopores for analyte detection, seePCT Publication No. WO2013/083983, incorporated herein by reference.

In some embodiments, the nanopore is an MspA or homolog or derivativethereof. MspA is formed from multiple monomers. The pore may behomomonomeric or heteromonomeric, where one or more of the monomerscontains a modification or difference from the others in the assemblednanopore. Descriptions of modifications to MspA nanopores have beendescribed, see U.S. Publication No. 2012/0055792, incorporated herein byreference in its entirety. Briefly described, MspA nanopores can bemodified with amino acid substitutions to result in a MspA mutant with amutation at position 93, a mutation at position 90, position 91, or bothpositions 90 and 91, and optionally one or more mutations at any of thefollowing amino acid positions: 88, 105, 108, 118, 134, or 139, withreference to the wild type amino acid sequence. In one specificembodiment, the MspA contains the mutations D90N/D91N/D93N, withreference to the wild type sequence positions (referred to therein as“M1MspA” or “M1-NNN”). In another embodiment, the MspA contains themutations D90N/D91N/D93N/D118R/D134R/E139K, with reference to the wildtype sequence positions (referred to therein as “M2MspA”). See U.S.Publication No. 2012/0055792. Such mutations can result in a MspAnanopore that comprises a vestibule having a length from about 2 toabout 9 nm and a diameter from about 2 to about 6 nm, and a constrictionzone having a length from about 0.3 to about 3 nm and a diameter fromabout 0.3 to about 3 nm, wherein the vestibule and constriction zonetogether define a tunnel. Furthermore, the amino acid substitutionsdescribed in these examples provide a greater net positive charge in thevestibule of the nanopore, further enhancing the energetic favorabilityof interacting with a negatively charged polymer analyte end.

Some nanopores, such as MspA protein nanopores, can comprise a variablyshaped tunnel component through which the polymer analyte moves. Forexample, an exemplary embodiment where MspA is disposed in a lipidbilayer membrane. The MspA nanopore comprises an outer entrance rimregion that contacts the illustrated enzyme. The widest interior sectionof the tunnel is often referred to as the vestibule. The narrowestportion of the interior tunnel is referred to as the constriction zone.The vestibule and a constriction zone together form the tunnel. A“vestibule” in MspA is a cone-shaped portion of the interior of thenanopore whose diameter generally decreases from one end to the otheralong a central axis, where the narrowest portion of the vestibule isconnected to the constriction zone. Stated otherwise, the vestibule ofMspA may generally be visualized as “goblet-shaped.” Because thevestibule is goblet-shaped, the diameter changes along the path of acentral axis, where the diameter is larger at one end than the oppositeend. The diameter may range from about 2 nm to about 6 nm. Optionally,the diameter is about, at least about, or at most about 2, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0 nm, or any rangederivable therein. The length of the central axis may range from about 2nm to about 6 nm. Optionally, the length is about, at least about, or atmost about 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or6.0 nm, or any range derivable therein. When referring to “diameter”herein, one can determine a diameter by measuring center-to-centerdistances or atomic surface-to-surface distances.

The term “constriction zone” generally refers to the narrowest portionof the tunnel of the nanopore, in terms of diameter, that is connectedto the vestibule. The length of the constriction zone can range, forexample, from about 0.3 nm to about 20 nm. Optionally, the length isabout, at most about, or at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or 3 nm, orany range derivable therein. The diameter of the constriction zone canrange from about 0.3 nm to about 2 nm. Optionally, the diameter isabout, at most about, or at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or 3 nm, orany range derivable therein. In other embodiment, such as thoseincorporating solid state pores, the range of dimension (length ordiameter) can extend up to about 20 nm. For example, the constrictionzone of a solid state nanopore is about, at most about, or at leastabout 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1, 2 13, 14, 15, 16,17, 18, 19, or 20 nm, or any range derivable therein. Larger dimensionin such nanopores can be preferable depending on the polymer used in themethod. As described in more detail below, the constriction zone isgenerally the part of the nanopore structure where the presence of apolymer, such as a nucleic acid, can influence the ionic current fromone side of the pore to the other side of the nanopore. FIG. 2B providesan illustrative diagram of a constriction zone that is sensitive to asubsequence of several nucleotides of a polymer. In this example, aspecific position within the constriction zone has the highestsensitivity for determining the current through the nanopore, asindicated by the vertical line and an indication of 0 nm displacement.Thus, the nucleotide residing in that position at any time will providethe greatest influence on the current signal and the neighboringnucleotides in the constriction zone have diminished influence on thesignal. Accordingly, the dimensions of the nanopore's constriction zonecan influence the resolution of the current signal as it relates to thestructure (and sequence identity) of the analyte polymer residingtherein. In some instances, the term “constriction zone” is used in afunctional context based on the obtained resolution of the nanopore and,thus, the term is not necessarily limited by any specific parameter ofphysical dimension. Thus, a nanopore's functional constriction zone canbe optimized by modifying aspects of the nanopore system but withoutproviding for any physical modification to the nanopore itself.

In some embodiments, the nanopore can be a solid state nanopore. Asolid-state layer is not of biological origin. In other words, a solidstate layer is not derived from or isolated from a biologicalenvironment such as an organism or cell, or a synthetically manufacturedversion of a biologically available structure. Solid state nanopores canbe produced as described in U.S. Pat. Nos. 7,258,838 and 7,504,058,incorporated herein by reference in their entireties. Briefly, solidstate layers can be formed from both organic and inorganic materialsincluding, but not limited to, microelectronic materials, insulatingmaterials such as Si3N4, Al203, and SiO, organic and inorganic polymerssuch as polyamide, plastics such as Teflon®, or elastomers such astwo-component addition-cure silicone rubber, and glasses. The solidstate layer may be formed from graphene. Suitable graphene layers aredisclosed in WO 20091035647 and WO 20111046706. Solid state nanoporeshave the advantage that they are more robust and stable. Furthermore,solid state nanopores can in some cases be multiplexed and batchfabricated in an efficient and cost-effective manner. Finally, theymight be combined with micro-electronic fabrication technology. In someembodiments, the nanopore comprises a hybrid protein/solid statenanopore in which a nanopore protein is incorporated into a solid statenanopore. In some embodiments, the nanopore is a biologically adaptedsolid-state pore.

In some cases, the nanopore is disposed within a membrane, thin film,layer, or bilayer. For example, biological (e.g., proteinaceous)nanopores can be inserted into an amphiphilic layer such as a biologicalmembrane, for example, a lipid bilayer. An amphiphilic layer is a layerformed from amphiphilic molecules, such as phospholipids, which haveboth hydrophilic and lipophilic properties. The amphiphilic layer can bea monolayer or a bilayer. The amphiphilic layer may be a co-blockpolymer. Alternatively, a biological pore may be inserted into a solidstate layer.

The membrane, thin film, layer, or bilayer typically separates a firstconductive liquid medium and a second conductive liquid medium toprovide a nonconductive barrier between the first conductive liquidmedium and the second conductive liquid medium. The nanopore, thus,provides liquid communication between the first and second conductiveliquid media through its internal tunnel. In some embodiments, the poreprovides the only liquid communication between the first and secondconductive liquid media. The conductive liquid media typically compriseselectrolytes or ions that can flow from the first conductive liquidmedium to the second conductive liquid medium through the interior ofthe nanopore. Liquids employable in methods described herein arewell-known in the art. Descriptions and examples of such media,including conductive liquid media, are provided in U.S. Pat. No.7,189,503, for example, which is incorporated herein by reference in itsentirety. The first and second liquid media may be the same ordifferent, and either one or both may comprise one or more of a salt, adetergent, or a buffer. Indeed, any liquid media described herein maycomprise one or more of a salt, a detergent, or a buffer. Additionally,any liquid medium described herein may comprise a viscosity alteringsubstance or a velocity altering substance.

In some cases, the first and second conductive liquid media located oneither side of the nanopore are referred to as being on the cis andtrans regions, where the protein analyte and the associated polymer areprovided in the cis region. However, it will be appreciated that in someembodiments, the protein analyte to be analyzed and the associatedpolymer can be provided in the trans region and, upon application of theelectrical potential, the polymer enters the nanopore from the transside of the system. In some cases, the entire length of the polymer doesnot pass through the pore, but only certain portions or segments of thepolymer pass through the nanopore for analysis. The directionality andrate of translocation can be regulated using various mechanisms such asapplied voltage or the incorporation of a nanopore in the reverseorientation.

Nanopore systems also incorporate structural elements to measure and/orapply an electrical potential across the nanopore-bearing membrane orfilm. For example, the system can include a pair of drive electrodesthat drive current through the nanopores. Typically, the negative poleis disposed in the cis region and the positive pole is disposed in thetrans region. Additionally, the system can include one or moremeasurement electrodes that measure the current through the nanopore.These can include, for example, a patch-clamp amplifier or a dataacquisition device. For example, nanopore systems can include anAxopatch-200B patch-clamp amplifier (Axon Instruments, Union City,Calif.) to apply voltage across the bilayer and measure the ioniccurrent flowing through the nanopore. For example, in some embodiments,the applied electrical field includes a direct or constant current thatis between about 10 mV and about 1 V. In some embodiments that includeprotein-based nanopores embedded in lipid membranes, the applied currentincludes a direct or constant current that is between about 10 mV and300 mV, such as about 10 mV, 20 mV, 30 mV, 40 mV, 50 mV, 60 mV, 70 mV,80 mV, 90 mV, 100 mV, 110 mV, 120 mV, 130 mV, 140 mV, 150 mV, 160 mV,170 mV, 180 mV, 190 mV, 200 mV, 210 mV, 220 mV, 230 mV, 240 mV, 250 mV,260 mV, 270 mV, 280 mV, 290 mV, 300 mV, or any voltage therein. In someembodiments, the applied electrical field is between about 40 mV andabout 200 mV. In some embodiments, the applied electrical field includesa direct or constant current that is between about 100 mV and about 200mV. In some embodiments, the applied electrical direct or constantcurrent field is about 180 mV. In other embodiments where solid statenanopores are used, the applied direct or constant current electricalfield can be in a similar range as described, up to as high as 1 V. Aswill be understood, the voltage range that can be used can depend on thetype of nanopore system being used and the desired effect.

Persons of skill in the art will readily appreciate that the reverseelectrical potential as the values and ranges described above can alsobe applied. This may be applicable where a molecular motor ischaracterized in the context of an electrical field that resists theforce applied by the molecular motor on the polymer.

In some embodiments, the electrical potential is not constant, butrather is variable about a reference potential. Such use of variablepotential in the context of a nucleic acid polymer can cause stretchingof the polymer to provide for more data sampling for each position ofthe polymer relative to the nanopore. This can be applied to methodsinvolving a molecular motor with a dynamic association with the polymer,or to methods involving covalently coupled polymers that do not move indiscrete steps but are rather anchored by the protein. SeePCT/US2014/059360, incorporated herein by reference in its entirety.

It is generally noted that the use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.”

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words “comprise,” “comprising,” and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to.” Words using the singular or pluralnumber also include the plural and singular number, respectively.Additionally, the words “herein,” “above,” and “below,” and words ofsimilar import, when used in this application, shall refer to thisapplication as a whole and not to any particular portions of theapplication. Words such as “about” and “approximately” imply minorvariation around the stated value, usually within a standard margin oferror, such as within 10% or 5% of the stated value.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. It is understoodthat, when combinations, subsets, interactions, groups, etc., of thesematerials are disclosed, each of various individual and collectivecombinations is specifically contemplated, even though specificreference to each and every single combination and permutation of thesecompounds may not be explicitly disclosed. This concept applies to allaspects of this disclosure including, but not limited to, steps in thedescribed methods. Thus, specific elements of any foregoing embodimentscan be combined or substituted for elements in other embodiments. Forexample, if there are a variety of additional steps that can beperformed, it is understood that each of these additional steps can beperformed with any specific method steps or combination of method stepsof the disclosed methods, and that each such combination or subset ofcombinations is specifically contemplated and should be considereddisclosed. Additionally, it is understood that the embodiments describedherein can be implemented using any suitable material such as thosedescribed elsewhere herein or as known in the art.

Publications cited herein and the subject matter for which they arecited are hereby specifically incorporated by reference in theirentireties.

The following describes an illustrative use of the disclosed method tocharacterize the association between the molecular motor, helicasehel308, with DNA at a sub-Angstrom level of resolution.

Abstract

The development of an in vitro high-resolution nanopore sensor toobserve enzyme activity is described. An electric field through theengineered protein porin, MspA, causes an ion current to flow. As theenzyme draws single stranded DNA through the pore, the nucleotides ofthe DNA control this ion current. Analysis of the ion current, providesa real-time record of how the enzyme processes the DNA. As demonstratedherein, the motion of DNA through enzymes can be resolved with up to 35pm longitudinal resolution and with sub-millisecond time scales.

The utility of this method on the helicase hel30 TGA by resolving anATP-dependent and an ATP-independent step for each single nucleotideadvance. The spatial and temporal resolution of this new low-cost singlemolecule technique allows exploration of hitherto unseen enzyme dynamicsin real-time.

Results and Discussion

A novel tool, referred to as Picometer Ioncurrent Nanopore Transducer(PINT), is presented here to examine molecular motors based on DNAtranslocation through a nanopore. PINT allows the observation of themotion of nucleic acids relative to the enzyme that processes them witha precision of tens of picometers, with a time scale shorter than amillisecond and with a load of 20-40 pN. PINT's intrinsic sensitivity isapplied herein by observing a helicase. In its basic form, PINT uses asingle nanometer-sized pore. An electrostatic potential applied acrossthe pore causes an ion current to flow. DNA, on which an enzyme isbound, is drawn into the pore by the electrostatic potential. The ssDNAfits through the pore but the enzyme is too wide to pass through thepore. Once the enzyme comes to rest on the pore, it limits the DNAtranslocation at the speed at which it the DNA moves through the enzyme,while the composition of the DNA in the narrowest part of the porecontrols the ion current. The ion current changes indicate the DNA'sprocession through the enzyme with surprisingly high precision (FIG. 2). In order for individual nucleotides to control the current, thenanopore must have features commensurate with the spacing between thenucleotides. Nature provides rugged protein pores with atomisticallyreproducible features. These pores can be customized through mutation.For our research in nanopore sequencing of DNA, we developed specificmutants (Butler, T. Z., et al., “Single-molecule DNA detection with anengineered MspA protein nanopore,” Proc. Natl. Acad. Sci. USA,105:20647-20652 (2008)) of the protein pore Mycobacterium smegmatisporin A, MspA. This pore has a short and narrow constriction (FIG. 1A)that is optimal for resolving individual nucleotides along DNA(Derrington, I. M., et al., “Nanopore DNA sequencing with MspA,” Proc.Natl. Acad. Sci. USA, 107:16060-16065 (2010); Manrao, E. A., et al.,PLoS ONE 6, e25723 (2011)). However, to realize MspA's high-resolutionsensing capability, DNA traversing through the pore must be heldstationary (Derrington, I. M., et al., Proc. Natl. Acad. Sci. USA,107:16060-16065 (2010); Manrao, E. A., et al., Nature Biotechnology 30:349-353 (2011)) or move slow enough to resolve picoampere currentchanges. In Manrao et al. (Manrao, E. A., et al., “Reading DNA atsingle-nucleotide resolution with a mutant MspA nanopore and phi29 DNApolymerase,” Nature Biotechnology 30:349-353 (2012)), we used the DNApolymerase (DNAP) of phi29 to draw DNA through MspA. We had observed asuccession of discrete ion current levels (FIG. 1B). The duration ofthese levels was stochastic with time constants of tens of milliseconds.Plotting the succession of ion current amplitudes revealed currentpatterns reproducible to within picoamperes (FIG. 1C). Each level wasassociated with the DNA's advance by one nucleotide and the currentpattern was matched to the DNA sequence (Manrao, E. A., et al., NatureBiotechnology 30:349-353 (2012)). In subsequent studies, we showed howthe magnitude of the ion current levels was related to the nucleotidesequence (Laszlo et al., “Decoding long nanopore sequencing reads ofnatural DNA,” Nature Biotechnology 32:829-833 (2014)), laying thefoundation of nanopore strand sequencing.

Because of the finite length of MspA's constriction zone and Brownianmotion, each current level was the time average involving about fournucleotides, effectively applying Gaussian smoothing to the successionof single nucleotide's current signals. Had the DNA been movedcontinuously, rather than in one nucleotide step, one would expect asmooth evolution of the ion current, i(x), where x is the position ofthe DNA relative to the pore. The discrete steps that the phi29 DNAPprovides sample this smooth curve at one-nucleotide intervals. Had theDNAP paused at additional steps that moved the DNA by a partialnucleotide, we would have observed additional levels at current valuesthat would fall on this smooth curve i(x). The position, x, of thesesteps can be found by inverting x=i⁻¹(x). Locally, a small positionchange Δx can be inferred from a current change Δi, in first order, byΔx=Δi/(di/dx). For DNA sequences that contain large di/dx, this allowsfor detection of DNA position changes of much less than one nucleotide.

To illustrate the achievable precision of PINT, we used a phi29 DNAP todraw a section of DNA through the pore's constriction that produces alarge and nearly linear slope in ion current. FIG. 2 shows the levelsaround an abasic site in DNA which produces particularly large currents.The superposition of levels from multiple DNA translocationsdemonstrates the reproducibility of levels. Assuming the current slopeto be linear and the interphosphate distance to be 690 pm, this resultsin only a ˜35 pm position uncertainty for a single passage of one DNAmolecule.

In another demonstration, we used PINT at driving forces of 180 mV and140 mV, again with DNA drawn by phi29 DNAP. Because of the elasticity ofthe ˜11 nucleotide-long DNA section between the DNAP and MspA'sconstriction, we expected that the level pattern at 140 mV would beshifted compared to that at 180 mV. After normalizing the currentamplitudes, we compared the two level patterns (FIG. 2E and FIG. 3D) andfound the patterns displaced by ˜0.3 nucleotide positions. This shift isin agreement with experimental force-stretching curves for ssDNA (Smithet al., Science, New Series, 271(5250):795-799 (1996); Bosco et al.,Nucleic Acids Research, 42(3) (2014)) demonstrating that the small DNAmotions are well resolved using PINT.

With PINT's precision established, we needed to demonstrate the tool'susefulness to study the molecular motors. Translocases, includinghelicases and polymerases, are particularly interesting for a variety ofreasons as they are associated with human aliments. For example,mutations in helicases are involved in a number of conditions, such asCerebro-oculo-facio-skeletal syndromes, Bloom, Werners andRothmund-Thomson, Baller-Gerold, and Warsaw Breakage syndromes, as wellas cancer. Mutations to human polymerases are also associated with anumber of abnormalities including mitochondrial diseases and cancers. Toensure their replication, viruses such as HIV, hepatitis C, and Ebolaencode their own helicases, polymerases, and/or packaging motors intheir genomes. Therefore, helicases and polymerases have becomepotential drug targets to interfere with viral infections andmechanistic understanding of these motors is particularly valuable.

Here we studied the superfamily II (SF2) helicase, hel308. He1308 is anATP-dependent ski2-like helicase/translocase that unwinds duplex DNAmoving on a single strand in the 3′ to 5′ direction. He1308 is found tobe conserved in many archaea as well eukaryota and is also found inhumans. With a known crystal structure, hel308 is a good model systemfor understanding processive SF2 enzymes. We chose the robust hel308 ofThermococcus gammatolerans EJ3 (Accession #YP_002959236.1).

We used similar or identical DNA constructs as in previously reportedDNA sequencing experiments with phi29 DNAP (Manrao, E. A., et al.,Nature Biotechnology 30:349-353 (2012), Laszlo et al., NatureBiotechnology 32:829-833 (2014)) (FIG. 2E), but in order to move theDNA's 5′ end towards MspA's vestibule, as during the enzymatic activityof the DNAP experiments, we annealed a complementary strand to our DNAsamples. When the single stranded 5′ end overhang filed through MspA'sconstriction, the complimentary strand was practically instantlyremoved, letting the DNA pass through the pore until it was held by thehelicase. With ATP present, the helicase began reeling the DNA backthrough the pore, ultimately returning it to the cis side. We recorded1000 current traces consistent with enzymatic activity. The currentpatterns were qualitatively similar to those observed with phi29 DNAP(FIG. 3A), but for hel308, we found approximately twice as many levels(FIG. 3B) as with phi29 DNAP, even though the same number nucleotideshad passed through the constriction. Similar results were obtained withother DNA sequences (not shown). We concluded that PINT's spatial andtemporal resolution allowed us to observe the internucleotide motion ofDNA through the helicase directly. After aligning to the known sequenceand current patterns (Laszlo et al., Nature Biotechnology 32:829-833(2014)), and building consensus current level patterns (FIG. 3A), weobserved that the average duration of levels alternated between long andshort levels. The duration distribution of each level is characterizedby its own time constant. FIG. 3C shows the mean duration for eachlevel, further elucidating that each one-nucleotide advance involves twodistinct steps with distinct time constants.

To investigate the origin of the two steps, we varied the ATPconcentration. FIG. 3D shows the average duration for the two stepsplotted against the ATP concentration (FIGS. 3C, 3D, and 3E). The ATPtitration reveals that one of the steps is ATP-dependent, while theother step is ATP-independent. Under the conditions of the experiment(22° C., 300 mM KCl, 5 mM MgCl₂, and 180 mV), the ATP-dependent stepfollowed Michaelis Menton kinetics with a maximum velocity of15.2+/−1.3/s and the Michaelis constant of 92.5+/−9.9 μmol. For theATP-independent step we measured a rate of 4.5+/−0.4 s⁻¹. FIG. 3Fsuggests that step duration may depend on sequence. We correlated thehalf-life of each step with the sequence offset by n_(off) nucleotides.We found that guanine 14 nucleotides from the constriction is associatedwith increased level duration (FIG. 3E) in the ATP independent step.This offset corresponds to a nucleotide position located well withinhel308.

Next, we analyzed the length of the two substeps along the DNA. We useda 3^(rd) order spline to model the smooth continuous current profilederived from the consensus of 393 translocation events. UsingATP-dependent levels, we interpolated the distance to theATP-independent level towards the 5′ side. The distribution of steplengths in units of nucleotide spacing were converted to distance alongthe DNA using the contour length Lc=690 pm/base (Bosco et al., NucleicAcids Research, 42(3) (2014)). Fitting the step length to theuncertainty weighted step size distribution yields a most probable valueof 0.43±0.11 L_(c).

It should be noted that the step length may depend on the location ofthe hel308 contact points with the rim of MspA.

The spatial and temporal resolution of this new single moleculetechnique allows exploration of hitherto unseen enzyme dynamics inreal-time. The unprecedented real-time resolution of PINT allowsobserving DNA motion of just a few tens of picometers. At first it maybe surprising that such a precision can be achieved given that Brownianmotion constantly repositions the enzyme on the pore, thereby affectingthe DNA's position in the constriction. However, Brownian motionexplores all possible configurations with such a high rate so that amillisecond measurement delivers a precise average value. It appearsthat much of the remaining spatial, as well as temporal fluctuationsobserved with PINT, can be attributed to the enzyme's activity. It islikely that the study of these fluctuations using PINT will contributeto detailed mechanistic understanding of the enzyme functioning.

Similar to many other single molecule techniques, PINT applies a forceof tens of piconewtons to the enzyme. In order to extrapolate to in vivoconditions, which are generally in a lower force regime, we calibratedthe force by comparing to DNA stretching curves taken with opticaltweezer (Smith et al., Science, New Series, 271(5250):795-799 (1996);Bosco et al., Nucleic Acids Research, 42(3) (2014)). At 180 mV, wherethe bulk of the PINT data was taken, the force on the DNA is estimatedto be 35±10 pN. A direct force measurement with reduced uncertainty isin progress.

PINT's extreme sensitivity can also be extended to other molecularmotors that do not process polymers. By attaching DNA to such enzymes ormotors, it will be possible to measure real-time conformational changesassociated with the enzyme activity.

TABLE 1 compares PINT with the most often used single moleculetechniques used to study translocase activity.

TABLE 1 Comparison of single molecule techniques to study translocases.Single molecule Spatial Temporal Through- technique resolutionresolution put Force Comments Example Reference(s) TIR smFRET <3 bp 30ms (8 ms) 200-400 0 pN Limited spatial (Myong et al., 2007) range 2-8 nmConfocal <3 bp 1 ms >100 0 pN No time trajectories (Theissen et al.,2008) smFRET of individual molecules Magnetic 10 bp 50 ms 10-20 5-40 pNForce method (Dessinges et al., 2004) tweezers throughput (Lionnet etal., 2007) with moderate (Sun et al., 2008) Optical Up to 1 bp 20 ms ~11-40 pN Many variants (Cheng et al., 2007) tweezers (Johnson et al.,2007) (Perkins et al., 2004) AFM force >5 bp 1 ms ~1 15-100 pN Lowthroughput; (Marsden et al., 2006) spectroscopy difficult cantileverfunctionalization PINT 0.05 bp <1 ms >100 10-40 pN Simple; The presentdisclosure inexpensive; sophisticated data analysis

PINT is highly parallelizable for high throughput desired in industrialapplications, such as drug screening. Importantly, PINT is a simple andlow-cost single molecule technique that can be practiced in a broadrange of laboratories.

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method ofcharacterizing a protein in a nanopore system comprising a nanoporedisposed in a membrane that separates a first conductive liquid mediumfrom a second conductive liquid medium, wherein the nanopore comprises atunnel that provides liquid communication between the first conductiveliquid medium and the second conductive liquid medium, and wherein theprotein is physically associated with a hydrocarbon polymer in the firstconductive liquid medium, wherein the hydrocarbon polymer comprises abackbone with a carbon chain, the method comprising: (a) applying anelectrical potential between the first conductive liquid medium and thesecond conductive liquid medium and causing the polymer to interact withthe nanopore tunnel, wherein the protein is unable to pass through thenanopore tunnel; (b) measuring an ion current through the nanoporeduring the interaction of the polymer with the nanopore tunnel toprovide a current pattern; (c) determining a position and/or movement ofat least one polymer subunit in the nanopore tunnel from the currentpattern; and (d) associating the position and/or movement of the atleast one polymer subunit with a characteristic of the protein.
 2. Themethod of claim 1, wherein the protein is an enzyme.
 3. The method ofclaim 2, wherein the enzyme is a molecular motor.
 4. The method of claim3, wherein the molecular motor is a translocase, a polymerase, ahelicase, an exonuclease, a viral packaging motor, or a topoisomerase.5. The method of claim 3, wherein the movement of the at least onepolymer subunit is associated with a length of a discrete translocationstep of the polymer within the nanopore tunnel that is conferred by themolecular motor.
 6. The method of claim 3, wherein the movement of theat least one polymer subunit is associated with a temporal duration of adiscrete translocation step of the polymer within the nanopore tunnelthat is conferred by the molecular motor.
 7. The method of claim 2,wherein the enzyme is a Brownian motor, Brownian ratchet ribosome,myosin, or kinesin.
 8. The method of claim 2, wherein the characteristicof the enzyme is a presence or degree of modulation of enzyme activityconferred by a reaction condition or putative agonist, antagonist, orco-factor.
 9. The method of claim 1, wherein the protein is a mutantprotein or fusion protein.
 10. The method of claim 1, wherein theprotein comprises two or more domains capable of mutual interaction. 11.The method of claim 1, wherein the protein is covalently coupled to thepolymer.
 12. The method of claim 1, wherein the position of the at leastone polymer subunit is associated with a conformational state of theprotein.
 13. The method of claim 1, wherein the nanopore is asolid-state nanopore, a protein nanopore, a hybrid solid state-proteinnanopore, a biologically adapted solid-state nanopore, or a DNA origaminanopore.
 14. The method of claim 13, wherein the protein nanopore isalpha-hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA),outer membrane porin F (OmpF), outer membrane porin G (OmpG), outermembrane phospholipase A, Neisseria autotransporter lipoprotein (NalP),WZA, Nocardia farcinica NfpA/NfpB cationic selective channel, lysenin ora homolog or variant thereof.
 15. A method of characterizing a proteinin a nanopore system comprising a nanopore disposed in a membrane thatseparates a first conductive liquid medium from a second conductiveliquid medium, wherein the nanopore comprises a tunnel that providesliquid communication between the first conductive liquid medium and thesecond conductive liquid medium, and wherein the protein is physicallyassociated with a hydrocarbon polymer in the first conductive liquidmedium, wherein the hydrocarbon polymer comprises a backbone with acarbon chain, the method comprising: (a) applying an electricalpotential between the first conductive liquid medium and the secondconductive liquid medium and causing the polymer to interact with thenanopore tunnel, wherein the protein is unable to pass through thenanopore tunnel; (b) measuring an ion current through the nanoporeduring the interaction of the polymer with the nanopore tunnel toprovide a first current pattern; (c) comparing the first current patternto a reference current pattern; (d) determining a change in positionand/or movement of at least one polymer subunit in the nanopore tunnelfrom the position and/or movement of at least one polymer subunit in thenanopore tunnel determined from the reference current pattern; and (e)associating the change in position and/or movement of the at least onepolymer subunit in the nanopore tunnel with a characteristic of theprotein.
 16. The method of claim 15, wherein the nanopore systemcomprises a difference from the nanopore system used to generate thereference current pattern.
 17. The method of claim 16, wherein thedifference is the presence, absence, or difference in concentration of aputative protein agonist, antagonist, or co-factor in the firstconductive medium.
 18. The method of claim 17, wherein thecharacteristic is a presence or degree of modulation of protein activityor conformation conferred by the putative agonist, antagonist, orco-factor.
 19. The method of claim 16, wherein the difference is atleast one amino acid difference in the amino acid sequence of theprotein compared to the amino acid protein sequence in the nanoporesystem used to generate the reference current pattern.
 20. The method ofclaim 19, wherein the characteristic is a presence or degree ofmodulation of protein activity or conformation conferred by the aminoacid difference in the amino acid sequence.